This invention is directed to a method for the generation of nucleic acid ligands having specific functions against target molecules using the SELEX process. The methods described herein enable nucleic acid ligands to be generated in dramatically shorter times and with much less operator intervention than was previously possible using prior art techniques. The invention includes a device capable of generating nucleic acid ligands with little or no operator intervention. The invention also includes reagents that can be used in any automated nucleic acid selection procedure to prevent the formation of high molecular weight amplification artifacts.
The dogma for many years was that nucleic acids had primarily an informational role. Through a method known as Systematic Evolution of Ligands by EXponential enrichment, termed the SELEX process, it has become clear that nucleic acids have three dimensional structural diversity not unlike proteins. The SELEX process is a method for the in vitro evolution of nucleic acid molecules with highly specific binding to target molecules and is described in U.S. patent application Ser. No. 07/536,428, filed Jun. 11, 1990, entitled xe2x80x9cSystematic Evolution of Ligands by EXponential Enrichment,xe2x80x9d now abandoned, U.S. Pat. No. 5,475,096 entitled xe2x80x9cNucleic Acid Ligandsxe2x80x9d, and U.S. Pat. No. 5,270,163 (see also WO 91/19813) entitled xe2x80x9cNucleic Acid Ligandsxe2x80x9d each of which is specifically incorporated by reference herein. Each of these patents and applications, collectively referred to herein as the SELEX Patent Applications, describes a fundamentally novel method for making a nucleic acid ligand to any desired target molecule. The SELEX process provides a class of products which are referred to as nucleic acid ligands or aptamers, each having a unique sequence, and which has the property of binding specifically to a desired target compound or molecule. Each SELEX-identified nucleic acid ligand is a specific ligand of a given target compound or molecule.
The SELEX process is based on the unique insight that nucleic acids have sufficient capacity for forming a variety of two- and three-dimensional structures and sufficient chemical versatility available within their monomers to act as ligands (form specific binding pairs) with virtually any chemical compound, whether monomeric or polymeric. Molecules of any size or composition can serve as targets. The SELEX method applied to the application of high affinity binding involves selection from a mixture of candidate oligonucleotides and step-wise iterations of binding, partitioning and amplification, using the same general selection scheme, to achieve virtually any desired criterion of binding affinity and selectivity. Starting from a mixture of nucleic acids, preferably comprising a segment of randomized sequence, the SELEX method includes steps of contacting the mixture with the target under conditions favorable for binding, partitioning unbound nucleic acids from those nucleic acids which have bound specifically to target molecules, dissociating the nucleic acid-target complexes, amplifying the nucleic acids dissociated from the nucleic acid-target complexes to yield a ligand-enriched mixture of nucleic acids, then reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield highly specific high affinity nucleic acid ligands to the target molecule.
It has been recognized by the present inventors that the SELEX method demonstrates that nucleic acids as chemical compounds can form a wide array of shapes, sizes and configurations, and are capable of a far broader repertoire of binding and other functions than those displayed by nucleic acids in biological systems. The present inventors have recognized that SELEX or SELEX-like processes could be used to identify nucleic acids which can facilitate any chosen reaction in a manner similar to that in which nucleic acid ligands can be identified for any given target. In theory, within a candidate mixture of approximately 1013 to 1018 nucleic acids, the present inventors postulate that at least one nucleic acid exists with the appropriate shape to facilitate each of a broad variety of physical and chemical interactions.
The basic SELEX method has been modified to achieve a number of specific objectives. For example, U.S. patent application Ser. No. 07/960,093, filed Oct. 14, 1992, now abandoned, and U.S. Pat. No. 5,707,796, both entitled xe2x80x9cMethod for Selecting Nucleic Acids on the Basis of Structure,xe2x80x9d describe the use of the SELEX process in conjunction with gel electrophoresis to select nucleic acid molecules with specific structural characteristics, such as bent DNA. U.S. patent application Ser. No. 08/123,935, filed Sep. 17, 1993, and U.S. patent application Ser. No. 08/443,959 filed May 18, 1995, both entitled xe2x80x9cPhotoselection of Nucleic Acid Ligands,xe2x80x9d and both now abandoned, and U.S. Pat. Nos. 5,763,177, U.S. patent application Ser. No. 6,001,577, WO 95/08003, U.S. Pat. No. 6,291,184, U.S. Pat. No. 6,458,539, and U.S. patent application Ser. No. 09/723,718, filed Nov. 28, 2000, each of which is entitled xe2x80x9cSystematic Evolution of Nucleic Acid Ligands by Exponential Enrichment: Photoselection of Nucleic Acid Ligands and Solution SELEX,xe2x80x9d all describe a SELEX process-based method for selecting nucleic acid ligands containing photoreactive groups capable of binding and/or photocrosslinking to and/or photoinactivating a target molecule. The resulting nucleic acid ligands are often referred to as xe2x80x9cphotoaptamers.xe2x80x9d These patents and patent applications are referred to in this application collectively as xe2x80x9cthe photoSELEX process applications.xe2x80x9d In the photoSELEX process variation of the SELEX process, a modified nucleotide activated by absorption of light is incorporated in place of a native base in either RNA- or in ssDNA-randomized oligonucleotide libraries.
U.S. Pat. No. 5,580,737 entitled xe2x80x9cHigh-Affinity Nucleic Acid Ligands That Discriminate Between Theophylline and Caffeine,xe2x80x9d describes a method for identifying highly specific nucleic acid ligands able to discriminate between closely related molecules, termed Counter-SELEX. U.S. Pat. No. 5,567,588 entitled xe2x80x9cSystematic Evolution of Ligands by EXponential Enrichment: Solution SELEX,xe2x80x9d describes a SELEX-based method which achieves highly efficient partitioning between oligonucleotides having high and low affinity for a target molecule. U.S. Pat. No. 5,496,938 entitled xe2x80x9cNucleic Acid Ligands to HIV-RT and HIV-1 Rev,xe2x80x9d describes methods for obtaining improved nucleic acid ligands after SELEX has been performed. U.S. Pat. No. 5,705,337 entitled xe2x80x9cSystematic Evolution of Ligands by Exponential Enrichment: Chemi-SELEX,xe2x80x9d describes methods for covalently linking a ligand to its target.
The SELEX method encompasses the identification of high-affinity nucleic acid ligands containing modified nucleotides conferring improved characteristics on the ligand, such as improved in vivo stability or improved delivery characteristics. Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base positions. SELEX process-identified nucleic acid ligands containing modified nucleotides are described in U.S. Pat. No. 5,660,985 entitled xe2x80x9cHigh Affinity Nucleic Acid Ligands Containing Modified Nucleotides,xe2x80x9d that describes oligonucleotides containing nucleotide derivatives chemically modified at the 5- and 2xe2x80x2-positions of pyrimidines. U.S. Pat. No. 5,580,737, supra, describes highly specific nucleic acid ligands containing one or more nucleotides modified with 2xe2x80x2-amino (2xe2x80x2-NH2), 2xe2x80x2-fluoro (2xe2x80x2-F), and/or 2xe2x80x2-O-methyl (2xe2x80x2-OMe). U.S. patent application Ser. No. 08/264,029, filed Jun. 22, 1994, entitled xe2x80x9cNovel Method of Preparation of Known and Novel 2xe2x80x2 Modified Nucleosides by Intramolecular Nucleophilic Displacement,xe2x80x9d describes oligonucleotides containing various 2xe2x80x2-modified pyrimidines.
The SELEX method encompasses combining selected oligonucleotides with other selected oligonucleotides and non-oligonucleotide functional units as described in U.S. Pat. No. 5,637,459 entitled xe2x80x9cSystematic Evolution of Ligands by EXponential Enrichment: Chimeric SELEX,xe2x80x9d and U.S. Pat. No. 5,683,867 entitled xe2x80x9cSystematic Evolution of Ligands by EXponential Enrichment: Blended SELEX,xe2x80x9d respectively. These applications allow the combination of the broad array of shapes and other properties, and the efficient amplification and replication properties, of oligonucleotides with the desirable properties of other molecules.
The SELEX method further encompasses combining selected nucleic acid ligands with lipophilic compounds or non-immunogenic, high molecular weight compounds in a diagnostic or therapeutic complex as described in U.S. Pat. No. 6,011,020 entitled xe2x80x9cNucleic Acid Ligand Complexesxe2x80x9d.
Nucleic acid ligands may be attached to the surface of solid supports to form microarrays. Such microarrays (also commonly referred to as xe2x80x9cbiochipsxe2x80x9d), and methods for their manufacture and use, are described in U.S. Pat. No. 6,242,246 U.S. patent application Ser. No. 08/211,680, filed December 14, 1998, now abandoned, WO 99/31275, U.S. Pat. No. 6,544,776, U.S. Pat. No. 6,503,715, and U.S. Pat. No. 6,458,543, each of which is entitled xe2x80x9cNucleic Acid Ligand Diagnostic Biochip.xe2x80x9d These patents are collectively as referred to herein as xe2x80x9cthe biochip applications.xe2x80x9d
One potential problem encountered in the diagnostic use of nucleic acids is that oligonucleotides in their phosphodiester form may be quickly degraded in body fluids by intracellular and extracellular enzymes such as endonucleases and exonucleases before the desired effect is manifest. Certain chemical modifications of the nucleic acid ligand can be made to increase the in vivo stability of the nucleic acid ligand or to enhance or to mediate the delivery of the nucleic acid ligand. See, e.g., U.S. patent application Ser. No. 08/117,991, filed Sep. 9, 1993, now abandoned, and U.S. Pat. No. 5,660,985, both entitled xe2x80x9cHigh Affinity Nucleic Acid Ligands Containing Modified Nucleotidesxe2x80x9d, and U.S. Pat. No. 6,387,620, entitled xe2x80x9cTranscription-free SELEXxe2x80x9d, each of which is specifically incorporated herein by reference. Modifications of the nucleic acid ligands contemplated in this invention include, but are not limited to, those which provide other chemical groups that incorporate additional charge, polarizability, hydrophobicity, hydrogen bonding, electrostatic interaction, and fluxionality to the nucleic acid ligand bases or to the nucleic acid ligand as a whole. Such modifications include, but are not limited to, 2xe2x80x2-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at exocyclic amines, substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil; backbone modifications, phosphorothioate or alkyl phosphate modifications, methylations, unusual base-pairing combinations such as the isobases isocytidine and isoguanidine and the like. Modifications can also include 3xe2x80x2 and 5xe2x80x2 modifications such as capping. In preferred embodiments of the instant invention, the nucleic acid ligands are DNA molecules that are modified with a photoreactive group on 5-position of pyrimidine residues. The modifications can be pre- or post-SELEX process modifications.
Each of the above described patent applications, many of which describe modifications of the basic SELEX procedure, are specifically incorporated by reference herein in their entirety.
Given the unique ability of the SELEX process to provide ligands for virtually any target molecule, it would be highly desirable to have an automated, high-throughput method for generating nucleic acid ligands.
The present invention includes methods and apparatus for the automated generation of nucleic acid ligands against virtually any target molecule. This process is termed the automated SELEX process. In its most basic embodiment, the method uses a robotic manipulator to move reagents to one or more work stations on a work surface where the individual steps of the SELEX process are performed. The individual steps include: 1) contacting the candidate nucleic acid ligands with the target molecule(s) of interest immobilized on a solid support; 2) partitioning the nucleic acid ligands that have interacted in the desired way with the target molecule on the solid support away from those nucleic acids that have failed to do so; and 3) amplifying the nucleic acid ligands that have interacted with the target molecule. Steps 1-3 are performed for the desired number of cycles by the automated SELEX process and apparatus; the resulting nucleic acid ligands are then isolated and purified.
Step 3 is performed with novel primers and candidate mixtures that minimize the formation of high molecular weight artifacts termed xe2x80x9cparasites.xe2x80x9d While not wishing to be bound by any one theory, it is believed that such parasites arise from rare mispriming events during the amplification process, and are propagated to subsequent rounds of the automated SELEX processxe2x80x94where they continue to grow in size and numberxe2x80x94unless they are size-fractionated from nucleic acid ligands at the conclusion of step 3. Size-fractionation procedures, such as gel electrophoresis, are time-consuming and difficult to automate. The primers and candidate mixtures provided herein drastically reduce the frequency of the intial mispriming event, and also drastically reduce the subsequent propagation of any rare parasites that do form. As a result, the automated SELEX process can be performed without size-fractionation procedures. The elimination of size-fractionation procedures greatly contributes to the utility, efficiency, and ease of operation of the automated SELEX process. Moreover, the techniques and procedures described herein and the primers and candidate mixtures provided by the instant invention will also be useful in any nucleic acid selection process that employs nucleic acid amplification without size-fractionation.
The automated SELEX process described herein enables the generation of large pools of nucleic acid ligands with little or no operator intervention. In particular, the methods provided by this invention will allow high affinity nucleic acid ligands to be generated routinely in just a few days, rather than over a period of weeks or even months as was previously required. The highly parallel nature of the automated SELEX process allows the simultaneous isolation of ligands against diverse targets in a single automated SELEX process experiment. Similarly, the automated SELEX process can be used to generate nucleic acid ligands against a single target using many different selection conditions in a single experiment. The present invention greatly enhances the power of the SELEX process, and will make SELEX the routine method for the isolation of ligands.